Our long-term goal is to develop strategies to obtain durable engraftment in allosensitized recipients. Although humoral immunity is important in solid organ grafting, the role of alloantibody (alloAbs) in bone marrow transplantation (BMT) has been largely ignored and difficult to treat. Because a single exposure to donor cells given 6 months pre-BMT can cause rapid humoral-mediated BM rejection at the time of BMT, long-lived antibody-secreting cells must be generated with the most likely candidates being plasma cells. Although much is known about Ab response to foreign antigens (Ags), the mechanisms responsible for alloAb generation have not been fully elucidated because of an inability to track alloAg-specific ASCs and possible differences in cell types that acquire, process and present alloAgs to the immune system. We propose to fill this knowledge gap to devise innovative strategies to overcome humoral-mediated rejection. Unless host T cell rejection can be subverted, overcoming humoral immunity will be inadequate to ensure engraftment. Despite the long held view that host T cells can reject donor bone marrow (BM), there is virtually no data as to the mechanisms responsible for alloprimed T cell mediated BM rejection. Memory T cells can reside in secondary lymphoid organs (SLOs), nonlymphoid tissues and in the BM. Due to technical difficulties, graft rejection studies have focused almost exclusively upon host T cells in SLOs and peripheral blood but SLOs may be dispensible for rejection in allosensitized recipients. Alloprimed host CTL responses to donor BM may differ in kinetics, magnitude or the requirement for donor BM antigen-presenting cells costimulation dependent upon their site of residence. Alloprimed graft rejecting T cells residing in host BM would not need to migrate from other sites. If so, interfering with host T cell migration to the BM would be ineffective. Using new models and tracking techniques, we will reevaluate existing rejection paradigms and use these data to design strategies to achieve engraftment in allosensitized hosts with both high titer alloAb and pre-existing alloprimed T cells. Our specific aims are: Aim 1: To define the mechanisms responsible for sustained, high titer alloAb and to devise strategies to overcome humoral-mediated rejection. Aim 2. To define the in vivo sites and dynamics of alloprimed T cell responses to donor BM that culminate in graft rejection and to devise strategies to overcome rejection in allosensitized recipients. Public Health Benefits Our goal is to develop clinically relevant approaches that will permit hematopoietic cell transplants in allosensitized patients. The fundamental insights gained from these studies will have broad implications relevant to both solid organ grafting and autoimmunity.